This invention relates generally to an amebocyte lysate preparation for use in the detection and/or quantification of a bacterial endotoxin in a sample, and more particularly to an endotoxin-specific amebocyte lysate preparation having reduced Factor G activity for use in the detection and/or quantification of a bacterial endotoxin in a sample.
Bacterial endotoxins, also known as pyrogens, are the fever-producing byproducts of Gram negative bacteria and can be dangerous or even deadly to humans. Symptoms of infection may range from fever, in mild cases, to death. In order to promptly initiate proper medical treatment, it is important to identify, as early as possible, the presence of an endotoxin and, if possible, the concentration of the endotoxin in the subject of interest. Similarly, the U.S. Food and Drug Administration (USFDA) requires certain manufacturers to establish that their products, for example, parenteral drugs and medical devices, are free of detectable levels of Gram negative bacterial endotoxin.
To this end, a variety of methods have been developed for use in the detection of bacterial endotoxins. A currently preferred method involves the use of amebocyte lysate (AL) produced from the hemolymph of a horseshoe crab, for example, a horseshoe crab selected from the group consisting of Limulus polyphemus, Tachpleus gigas, Tachypleus tridentatus, and Carcinoscorpius rotundicauda. Amebocyte lysates produced from Limulus, Tachpleus, and Carcinoscorpius maybe referred to as LAL, TAL, and CAL, respectively.
Presently, LAL is employed in bacterial endotoxin assays of choice because of its sensitivity, specificity and relative ease for avoiding interference by other components that may be present in a sample of interest LAL, when combined with a sample containing bacterial endotoxin, reacts with the endotoxin to produce a product, for example, a gel or chromogenic product, that can be detected, for example, either visually or by the use of an optical detector.
The endotoxin-mediated activation of LAL is well understood and has been thoroughly documented in the art. See, for example, Levin et at. (1968) Thromb. Diath. Haemorrh. 19: 186, Nakamura et al. (1986) Eur. J. Biochem. 154: 511, Muta et al. (1987) J. Biochem. 101: 1321, and Ho et al. (1993) Biochem. and Mol. Biol. Int. 29: 687. When bacterial endotoxin is contacted with LAL, the endotoxin initiates a series of enzymatic reactions, referred to in the art as the Factor C pathway, that involve at least three serine protease zymogens called Factor C, Factor B and pro-clotting enzyme (see FIG. 1). Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor, Factor C is activated. Activated Factor C thereafter hydrolyses and activates Factor B, whereupon activated Factor B activates proclotting enzyme to produce clotting enzyme. The clotting enzyme thereafter hydrolyzes specific sites, for example, Arg18-Thr19 and Arg46-Gly47 of coagulogen, an invertebrate, fibrinogen-like clottable protein, to produce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.
Although the clotting cascade of LAL initially was considered specific for endotoxin, it was later discovered that (1xe2x86x923)-B-D glucans also activate the clotting cascade of LAL through a unique enzymatic pathway, referred to in the art as the Factor G pathway (see FIG. 1). Upon exposure to (1xe2x86x923)-B-D glucan, Factor G is activated to produce activated Factor G. Activated Factor G thereafter converts the proclotting enzyme into clotting enzyme, whereupon the clotting enzyme converts coagulogen into coagulin, similar to the case with endotoxin. Accordingly, the coagulation system of LAL, like the mammalian blood coagulation system, consists of at least two coagulation cascades which include an endotoxin-mediated pathway (the Factor C pathway), and a (1xe2x86x923)-B-D glucan-mediated pathway (the Factor G pathway). See, for example, Morita et al. (1981) FEBS Lett. 129: 318-321 and Iwanaga et al. (1986) J. Protein Chem. 5: 255-268.
In view of the Factor C and Factor G pathways of LAL, the detection of bacterial endotoxin in a sample can, under certain circumstances, become ambiguous. As a result, attempts have been made to increase the specificity of LAL for endotoxin, i.e., to produce an endotoxin-specific amebocyte lysate preparation.
In one approach, polysaccharide based Factor G inhibitors are combined with amebocyte lysate to reduce or eliminate clotting induced by (1xe2x86x923)-B-D glucan present in the biological sample, i.e., inhibit the Factor G cascade. See, for example, U.S. Pat. Nos.: 5,155,032; 5,179,006; 5,318,893; 5,474,984; and 5,641,643.
In an alternative approach, several groups have attempted to remove Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate that is insensitive to (1xe2x86x923)-B-D glucan. For example, Obayashi et al. (1985) Clin. Chim. Acta 149:55-65 disclose a method for fractionating coagulation enzymes in LAL and then recombining only those factors involved in the endotoxin induced coagulation cascade (i.e., the Factor C cascade) to produce a Factor G depleted amebocyte lysate. The resulting lysate, however, may not only lack Factor G but also other components required for a complete Factor C cascade. The reconstituted lysate produced by this procedure, apparently does not produce a natural coagulin type clot and can be used only with synthetic chromogenic substrates.
U.S. Pat. No. 5,401,647 discloses a method for removing Factor G from LAL by combining LAL with (1xe2x86x923)-B-D glucan immobilized on an insoluble carrier. Once bound to the carrier via the (1xe2x86x923)-B-D glucan moiety, the Factor G can thereafter be removed from the LAL to produce a Factor G depleted lysate. Similarly, U.S. Pat. No. 5,605,806 discloses an immunoaffinity based method using a Factor G specific antibody to remove Factor G from LAL thereby to produce a Factor G depleted amebocyte lysate.
There still exists, however, a demand for an endotoxin-specific amebocyte lysate that can be produced economically in commercial quantities. A method for producing such an amebocyte lysate should be rapid, reproducible, inexpensive, simple to conduct, and preferably should result in an amebocyte lysate that can be used in a reliable, and quantitative determination of endotoxin in a sample of interest.
The invention features improved amebocyte lysate preparations having reduced Factor G activity, methods of making such lysate preparations, and methods of using such lysate preparations in the detection and/or quantitation of one or more bacterial endotoxins in a sample of interest.
In one aspect, the invention provides a method of producing an endotoxin-specific amebocyte lysate preparation for use in the detection of bacterial endotoxins in a sample. The amebocyte lysate preparation is rendered endotoxin-specific by the reduction and/or elimination of Factor G activity in the preparation. The amebocyte lysate preparation of the invention is produced by (a) admixing crude amebocyte lysate, i.e., amebocyte lysate reactive with both endotoxin and (1xe2x86x923)-B-D glucan, with a surfactant in an amount sufficient to produce a solution containing a precipitate; and (b) separating the precipitate from the solution thereby to produce an amebocyte lysate preparation which is less reactive with a (1xe2x86x923)-xcex2-D glucan than is the crude amebocyte lysate. The precipitate produced by addition of surfactant to crude lysate may contain any component necessary for a complete Factor G cascade, however, the production of a precipitate actually containing Factor G is preferred.
The amebocyte lysate preparation produced by the methodologies described herein comprises all the components necessary for a complete Factor C cascade, i.e., is still capable of producing a coagulin gel via the endotoxin-mediated pathway. Accordingly, the resulting amebocyte lysate preparation is capable of reacting with a bacterial endotoxin, e.g., a bacterial endotoxin produced by Gram negative bacteria, to produce a coagulin clot.
It is contemplated that any surfactant (otherwise known as a surface active agent or detergent) which produces a precipitate when added to crude amebocyte lysate, wherein the precipitate once removed from the lysate results in a reduction of Factor G activity, may be used in the practice of the invention. The surfactant, however, preferably is a zwitterionic surfactant, i.e., a surfactant having a headgroup containing both a negatively charged chemical moiety and a positively charged chemical moiety. Examples of zwitterionic surfactants include betaines and sulfobetaines, however, sulfobetaine-type surfactants are preferred. Preferred sulfobetaine-type surfactants include, without limitation, n-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; and n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate. The sulfobetaine-type surfactant n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, however, is most preferred.
In one embodiment, the method comprises the additional step of removing from or otherwise reducing the concentration of the added surfactant in the solution. The surfactant may be removed, for example, by chromatographic separation using, for example, a suitable ion exchange resin or, alternatively, by any other means known in the art for removing a particular surfactant from an aqueous solution. In a preferred method, the surfactant is removed by conventional organic solvent extraction. Any organic solvent that dissolves the surfactant of interest and is compatible with amebocyte lysate may be used in the solvent extraction step, however, for the reasons discussed below, chloroform is preferred.
In another embodiment, the sensitivity to bacterial endotoxin of an amebocyte lysate preparation having reduced Factor G activity can be enhanced by the addition of exogenous (1xe2x86x923)-xcex2-D glucan to the amebocyte lysate preparation. In particular, (1xe2x86x923)-xcex2-D glucan is added to the lysate preparation in an amount sufficient to enhance the sensitivity of the lysate preparation to endotoxin relative to a similar amebocyte lysate preparation without exogenously added (1xe2x86x923)-xcex2-D glucan. Without wishing to be bound by theory, it appears that exogenously added (1xe2x86x923)-xcex2-D glucan acts synergistically with the endotoxin mediated pathway. It is understood, however, that the same amount of (1xe2x86x923)-xcex2-D glucan when added to crude amebocyte lysate, i.e., amebocyte lysate that is reactive with both endotoxin and (1xe2x86x923)-xcex2-D glucan, likely would induce the production of a coagulin gel via the Factor G cascade. In effect, during the practice of this particular embodiment of the invention, a substrate or initiator of the Factor G cascade is added to the amebocyte lysate preparation of the invention.
Although it is contemplated that any amount of (1xe2x86x923)-xcex2-D glucan that enhances the sensitivity of the amebocyte lysate to the endotoxin relative to similar amebocyte lysate without the exogenously added (1xe2x86x923)-xcex2-D glucan may be used in the practice of the invention, the optimal amount of exogenous (1xe2x86x923)-xcex2-D glucan for enhancing the endotoxin-specific cascade in a particular lysate can be determined by routine experimentation. For example, the optimal concentration can be determined by adding different amounts of a particular (1xe2x86x923)-xcex2-D glucan to crude amebocyte lysate, i.e., amebocyte lysate reactive with both endotoxin and (1xe2x86x923)-xcex2-D glucan. The optimal amount of the (1xe2x86x923)-xcex2-D glucan to be added to the amebocyte lysate preparation of the invention, can be determined using, for example, a kinetic turbidimetric assay whereby the optimal amount is the amount of (1xe2x86x923)-xcex2-D glucan that induces the fastest coagulin clot formation in crude amebocyte lysate. This assay protocol is exemplary, and it is understood that the skilled artisan may use a variety of other assays, for example, a gel-clot assay, an end-point turbidimetric assay, or a chromogenic assay, to determine the optimal amount of (1xe2x86x923)-xcex2-D glucan to be added to the lysate of interest.
It is contemplated that any (1xe2x86x923)-xcex2-D glucan that induces the Factor G cascade in crude amebocyte lysate can be used to enhance the sensitivity of the endotoxin mediated pathway in the amebocyte lysate preparation of the invention. Preferred (1xe2x86x923)-xcex2-D glucans include, without limitation, cotton extract; rinses from cellulose acetate membranes; curdlan; pachyman; scleratan; leutinan; schizophyllan; coriolan; laminaran; and laminarin. Laminarin, however, currently is most preferred.
In another aspect, the invention provides an amebocyte lysate preparation having reduced Factor G activity, i.e., an amebocyte lysate having reduced reactivity to (1xe2x86x923)-xcex2-D glucans relative to crude lysate produced by the aforementioned methodologies. In one embodiment, such a composition may comprise (i) an amebocyte lysate preparation having reduced Factor G activity or, most preferably, an amebocyte lysate preparation depleted of Factor G activity, and (ii) exogenously added (1xe2x86x923)-xcex2-D glucan, wherein the (1xe2x86x923)-xcex2-D glucan is added in an amount sufficient to enhance the sensitivity of the amebocyte lysate preparation to endotoxin relative to a similar amebocyte lysate preparation without the exogenously added (1xe2x86x923)-xcex2-D glucan. Determination of the optimal amount of a particular (1xe2x86x923)-xcex2-D glucan for enhancing sensitivity of the lysate to endotoxin has been discussed previously.
In another aspect, the invention provides methods for detecting and/or quantitating the amount of a bacterial endotoxin in a sample. The improvement in such methods resides in the use of the amebocyte lysate preparation of the invention.
The foregoing and other objects, features and advantages of the present invention will be made more apparent from the following detailed description of preferred embodiments of the invention.